Potentiators of insulin secretion

ABSTRACT

The present invention relates to a recognition that an analog of αKG can increase glucose-induced insulin secretion in vitro and in vivo in animals, particularly in mammals, and more particularly in humans and in rodents. By employing the methods of the invention, insulin secretion can be increased.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/922,442, filed Aug. 20, 2004, which claims the benefit of U.S.Provisional Application No. 60/496,802, filed Aug. 21, 2003, both ofwhich are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agency: NIH DK58037. The United States government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Pancreatic β cells secrete insulin in response to elevated amounts ofblood glucose and other blood borne metabolites termed “secretagogues.”Glucose is delivered to body tissues in part via transporters thatrespond to insulin. Insulin reduces blood glucose by stimulating itsremoval from the bloodstream and by inhibiting glucose production by theliver. In addition to glucose, other molecules stimulate β-cells tosecrete insulin. These include amino acids, α-ketoacids andmitochondrial metabolites. Collectively, these secondary secretagoguescan account for as much as 50% of all insulin secreted.

The control of blood glucose is initiated by Glut2-dependent glucoseuptake by pancreatic β-cells. Inside the cell, glucose is metabolized toyield ATP, which causes ATP-sensitive K⁺ ion channels (K_(ATP)) toclose, blocking the efflux of K⁺ ions and depolarizing the membranepotential. This depolarization opens voltage-gated Ca²⁺ ion channels(V_(Ca)), leading to a rise in intracellular Ca²⁺, which triggersvesicle mobilization and insulin secretion.

A genetically inherited condition characterized by higher than normalglutamate dehydrogenase enzyme activity arises from any of severalmutations at the site of GTP-mediated suppression of the enzyme. Theenzyme catalyzes oxidative glutamate deamination and produces ammonia,α-ketoglutarate (αKG), and reducing equivalents. Humans having thecondition exhibit hyperammonemia, hyperinsulinemia, and hypoglycemia.The mechanism by which excessive glutamate dehydrogenase activityincreases insulin secretion is not known. The conventional wisdom isthat stimulation of insulin release requires metabolism of αKG.

αKG is a Krebs cycle intermediate that can be oxidatively decarboxylatedto form succinate, an insulin secretagogue. αKG is also a stoichiometriccofactor of various αKG-dependent hydroxylase enzymes, includingprolyl-4-hydroxylases which have a variety of substrates, the beststudied being collagen. Prolyl-4-hydroxylases also regulatehydroxylation at a single proline residue on Hypoxia Inducible Factor-1α(HIF-1α), a transcription factor that regulates a potassium ATP(K_(ATP)) channel involved in insulin secretion from β cells.

Cellular αKG is formed by glutamate dehydrogenase (GDH) and in thecitric acid cycle via isocitrate dehydrogenase as well as by thebranched chain aminotransferase (BCAT) reaction wherein no reducingequivalents are produced. In this reaction, an α-ketoacid and an α-aminoacid are interconverted into their corresponding amino- and α-ketoacids.α-ketoisocaproic acid (KIC) is transaminated using an amine group fromglutamate to form leucine. In the process, glutamate loses an aminegroup and forms αKG. KIC-induced hypersecretion of insulin is preventedby blocking the transamination of KIC (using, e.g., BCAT inhibitormethyl-leucine) to leucine and the attendant formation of αKG. αKGformation may be part of a signaling cascade that leads to chronicinsulin hypersecretion. When applied to isolated pancreatic β-cells, KICis known to cause depolarization of the cell membrane voltage, leadingto generation of action potentials, increase in cytosolic Ca²⁺ ionconcentration and increased insulin secretion. KIC-dependentdepolarization is known to be due to a direct inhibition of K_(ATP). Inintact β-cells the flow of K⁺ ions through K_(ATP) is likely the currentthat dominates the resting membrane potential. Accordingly, agents thatmodulate K_(ATP) channel activity will necessarily alter membranepotential.

Persaud, S. J. et al., J. Molec. Endocrin. 22:19-28 (1999) disclosesthat protein tyrosine kinase inhibitors genistein and tyrphostin A47inhibited KIC-stimulated insulin release without affecting glucosemetabolism. Persaud et al. suggested that the protein tyrosine kinaseinhibitors exert their inhibitory effects distal to closure ofATP-sensitive K⁺ channels, but proximal to Ca²⁺ entry into β cells, andthat the inhibitors act at the site of the voltage-dependent Ca²⁺channel that regulates Ca²⁺ influx into β cells followingdepolarization.

A recently discovered heritable genetic disorder attributable tomutations in the GDH enzyme highlights the physiological importance ofαKG-dependent insulin segretagogue activity. The enzyme catalyzesconversion of glutamate to αKG and ammonia. In mutated form, the enzymecauses chronic hyper-insulinemia resulting in severe hypoglycemia.Interestingly, the mutations led to an increase in enzyme activity andover-production of αKG.

It would be advantageous to identify compounds that augment the amountof insulin secretion evoked by glucose and other secretagogues. Suchcompounds would have therapeutic utility in treating those forms ofdiabetes caused by insufficient β cell responsiveness to insulinsecretagogues.

BRIEF SUMMARY OF THE INVENTION

It is herein proposed for the first time that analogs of αKG canpotentiate insulin secretion in animals, particularly in mammals, andmore particularly in humans and in rodents. Rodents, particularly mice,provide a standard, art-accepted model system for studying onset andprogression of Type II diabetes, such as also occurs in humans. It isherein proposed and demonstrated that αKG and structural analogs thereofdirectly regulate the activity of K_(ATP) ion channels and therebyprovide additional regulation of insulin secretion by enhancing theglucose-dependent regulation of K_(ATP). In a related aspect, therefore,the invention finds particular applicability to treatment ofhyperglycemia in individuals having Type II diabetes in a method thatcomprises the steps of administering to such an individual an effectiveamount of a suitable structural analog of αKG as described herein andobserving an increase in insulin secretion from pancreatic β cells.

The invention is further summarized in that a preferred αKG analog foruse in the methods of the invention is 2,4-diethyl pyridinedicarboxylate (DPD), also known as diethyl-2-4-pyridine dicarboxylate, aknown and commercially available inhibitor of prolyl-4-hydroxylaseactivity.

A skilled artisan can arrive at still further solutions for achievingthe goals of the methods by selecting and testing putative agents foreffectiveness in the methods. It will also be understood that thecompound can be administered in a preparation that comprises a pluralityof agents, where at least one agent contributes to the overallinsulin-controlling effect.

It is an object of the present invention to increase insulin secretionin individuals having hyperglycemia.

It is an advantage of the present invention that the K_(ATP) channel isan accessible and safe target for drug development in this disease area,insofar as the SUR1 regulatory subunit is a known target forsulfonylurea drugs for treating hyperglycemia.

It is another advantage of the present invention that the naturalactivity of αKG can be effectively mimicked by compounds having suitablestructures described herein. The compounds are not subject to the samemetabolic processes in vivo as αKG and are therefore not cleared fromthe system in the same manner or at as rapid a rate.

It is yet another advantage of the present invention that the αKGanalogs enhance, or strengthen, glucose-dependent regulation of K_(ATP),stimulating greater insulin secretion at high rather than low glucoseconcentrations, and thereby reducing the risk of inducing hypoglycemia,a critical liability associated with the sulfonylurea drugs.

It is yet another advantage of the present invention to provide methodsfor improving insulin secretion in response to glucose and othersecretagogues in individuals having inadequate response to these agents.

Other objects, advantages, and features of the invention will becomeapparent upon consideration of the following detailed description takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts the ability of various analogs of DPD to increase insulinsecretion from pancreatic β cells.

FIG. 2 illustrates an active pharmacophore for an αKG analog thatfunctions as an insulin segretagogue.

FIG. 3 compares potentiation by αKG and DPD of insulin release fromhyperexcitable Newgard Ins-1 cells under low or high glucose conditions.

FIG. 4 compares insulin release from two different β cell lines, namelyhyperexcitable Newgard Ins-1 cells and standard Ins-1 cells.

DETAILED DESCRIPTION OF THE INVENTION

The physiological relevance of insulin secretion from pancreatic β cellselicited by αKG was evaluated. Insulin secretion caused by αKG and DPD,a non-metabolizable analog of αKG, was blocked when the influx of Ca²⁺ions was suppressed by either bathing cells in Ca²⁺-free medium or byusing an L-type Ca²⁺ channel antagonist, nitrendipine. Further,application of α₂-adrenergic agonists epinephrine or clonidine greatlysuppressed insulin secretion caused by glucose, KIC, αKG or DPD. Takentogether, these observations suggest that αKG promotes insulin releaseutilizing the normal physiological mechanisms of triggered insulinsecretion.

Possible targets for the segretagogue action of αKG were examined byreevaluating targets implicated for KIC-dependent increases in insulinsecretion, since the effects of KIC are likely exerted throughtransamination that yields αKG. The inventors hypothesized that αKG, DPDand its analogs stimulate increased insulin release by modulatingK_(ATP) directly, since it is believed that the KIC-dependent effect oninsulin secretion requires transamination of KIC to yield αKG. Tobiochemically test this hypothesis the inventors used known drugs tomodulate K_(ATP) and then determined whether αKG and DPD could stillaffect insulin secretion. Diazoxide, a K_(ATP) agonist, completelysuppresses the ability of αKG, DPD and KIC to promote insulin release.However, glyburide, a K_(ATP) antagonist, elicits an insulin secretionthat is augmented by αKG, DPD or KIC. These results suggest that αKG andDPD inhibit the flow of K⁺ ions through K_(ATP), leading to membranedepolarization, entry of Ca²⁺ ions and insulin release. In addition, αKGand DPD appear to target a K_(ATP)-independent process similar to thatshown for glucose.

DPD and structurally related compounds (as disclosed below) that yieldmore insulin secretion from cells than are preferred agents for use inthe methods of the invention. DPD is extremely potent relative to αKGand greatly amplifies the stimulatory effect of glucose on insulinsecretion. DPD and structurally related compounds can be used to treatdiabetes, particularly when used at a concentration at which thecompound does not elicit significant insulin release in the absence ofan elevated blood glucose concentration, thereby reducing the risk ofhypoglycemia, a problem encountered with the current sulfonylureamedications. In the methods, the agent is formulated with apharmaceutically acceptable carrier for bioavailability to pancreatic βcells and is administered in an amount sufficient to inhibit KIC-inducedinsulin secretion in an animal. The compound can be administered byvarious administration routes including, but not limited to, oral,intravenous, subcutaneous and intraperitoneal routes. The compoundshould be administered in an amount sufficient to give rise to aconcentration of the compound in plasma of between about 0.1 to 1.0 mM.

The method of the invention is considered effective if, after the agentis administered, an increase in insulin comparable to the increasesobserved using available glyburide compounds (e.g., 47% for repaglinide,54% for glipizide and 61% for glybenclamide, as reported in DiabetesCare 25:1271 (2002), incorporated herein by reference as if set forth inits entirety). Accordingly an increase of at least about 1.25 fold toless than 3 fold is preferably observed relative to the pretreatmentplasma insulin level. Alternatively, and especially in non-human modelanimals, the effectiveness of the treatment can be tested by comparingfreshly explanted pancreatic islet cells that were treated in vivo withlike cells explanted from an untreated control to determine whetherinsulin secretion is increased. Methods for culturing pancreatic isletcells are known in the art.

The ability of various structurally-related compounds to alter insulinsecretion in the presence of high glucose was tested in a preliminarystructure-activity relationship (SAR) study to elucidate the molecularstructures relevant to the insulin segretagogue potential of DPD andother αKG analogs. The compounds tested, shown in FIG. 1, are presentedin order of decreasing ability to secrete insulin in an Ins-1 cell assayof the type described below in the Working Example. Each tested compoundis commercially available and all were purchased from Sigma-Aldrich.Each compound contained two carboxylate groups. Variables evaluated inthis study included the relative position of the carboxylate groups toone another, as well as the presence or absence of a nitrogen or sulfurgroup, additional R-groups, and esterified carboxy groups.

The preliminary study revealed that:

1. Compounds 2 and 6 were more potent than DPD, eliciting ˜31% and 25%secretion respectively, while DPD caused ˜14% secretion. Thisdemonstrates that analog compounds having at least twice the potency ofDPD can be employed in the methods of the invention.

2. a suitable αKG analog does not require an aromatic nitrogen, asCompound 8 and DPD were equipotent.

3. a suitable αKG analog preferably has a generally planar geometryimposed by an aromatic ring structure, as Compounds 1, 2 and 4 sharevery similar structures, but the hydrogen group attached to the aromaticnitrogen of inactive Compound 4 destabilizes the planar geometry. Thissuggests a preferred positioning of the carboxylate groups in a sharedplane.

4. in a suitable αKG analog, the carboxylate groups are preferablyseparated by 3 carbon atoms, as the highest insulin secretion activityis observed using compounds having 3 carbons between the carboxylategroups. Two-carbon separation, as in the case of active Compound 9, orfour-carbon separation, as in the case of pyridine-2,5-dicarboxylate canalso be acceptable.

5. in a suitable αKG analog, the carboxylate groups are protected byester groups (or alternatively and less preferably by thioesters oramides). This observation derives from comparing the potency of the mostpotent Compound 2, which has an ethyl ester group protecting eachcarboxylate group, and one of the least potent compounds (Compound 12),which differs from Compound 2 only in that it lacks the ethyl estergroups. Because the negative charge associated with un-protectedcarboxylate groups would prevent Compound 12 from crossing the cellularmembrane, this result, in combination with the above-noted observationthat diazoxide suppresses insulin secretion by αKG and DPD, suggeststhat Compound 12 cannot reach a target on the cell interior and that thesuitable analogs bind on the inner surface of the cell membrane,particularly to surface-bound K_(ATP).

6. a suitable αKG analog can, but need not comprise an exocyclic aminegroup, although such a group can be preferred in that it offers aconjugation “handle” for amine-reactive probes such as, but not limitedto, a fluorescent or photo-labile group of the sort commonly used toprobe binding interactions between a small molecule and its target. Forexample, Compound 6 is very active and contains an exocyclic aminegroup.

Structurally, an αKG analog that can function as an insulin segretagoguehas a ring core, two carboxyl groups or ester derivatives thereofattached to one or preferably two separate ring atoms, and optionallyother side groups. The ring core is preferably a 3- to 6-membered ringand more preferably a 5- or 6-membered aromatic ring. The ring cancontain heteroatoms (i.e., non-carbon atoms) such as nitrogen, sulfur,and oxygen atoms. Nitrogen is the preferred heteroatom. The two carboxylgroups or ester derivatives thereof attached to the ring are preferablyseparated by at least three ring atoms including the two to which theyare attached.

Incorporating these observations into a structural model, FIG. 2illustrates an active pharmacophore for an αKG analog that functions asan insulin segretagogue. The skilled artisan will appreciate the rangeof compounds having this structure. A systematic process of evaluatingthe activity of compounds having this active pharmacophore may lead to amore refined pharmacophore. Small groups (e.g., H or CH₃) are preferredat R2. In the structural ring that includes R4, R5 and R6, an aromaticfive- or six-membered ring is preferred, although larger rings can beemployed (n at R4 can be greater than greater than or equal to 1). Asix-membered ring can contain 3 double bonds; a five-membered ring cancontain a heteroatom (N, O or S) and two double bonds. The heteroatomcan be at R4 or at R5 if R6 does not exist. R1 and R3 are alsopreferably small groups on the order of 1-4 carbons. It will also beappreciated that if a thioester or amide protecting group is employed,the oxygens bound to R1 and R3 would be replaced with sulfur ornitrogen, respectively.

Among the αKG analogs evaluated, DPD is considered a preferred analogbecause it mimics the stimulation of insulin secretion observed with αKGbut is not metabolized like αKG in vivo. Other compounds, especiallycompounds that cannot be similarly metabolized, having still higheractivity can be considered to be still more preferable. Further, if αKGand DPD are applied together at subsaturation levels, the resultinginsulin secretion is less than the sum of either compound added alone,suggesting that αKG and DPD impact the same target to promote insulinrelease. Metabolism of αKG is not required for insulin secretion,although αKG production appears to be required for insulin segretagogueactivity under the tested conditions.

The present invention will be more fully understood upon considerationof the following non-limiting examples, performed in vitro in glucosecultures of freshly explanted pancreatic islets cultured with KIC in thepresence or absence of the exemplified analog and in cultured cells.

WORKING EXAMPLE

Mouse islets from diabetes-resistant (C57BL/6) or diabetes-susceptible(BTBR) mice were isolated by collagenase digestion, 0.45 mg/mlcollagenase (Sigma, Type XI collagenase) in Hanks Balanced Salt Solution(HBSS) containing 0.02% BSA, 0.35g/L NaHCO3. After two washes in HBSSwithout collagenase, islets were separated from digested acinar tissueby centrifugation on Ficoll gradients. Islets were then further purifiedby picking under a dissection microscope.

Insulin released from freshly isolated islets was measured in a staticincubation system. Briefly, islets were hand picked with great care toobtain five similar size islets per batch. Minimum of three batches offive islets were used for each incubation condition. Islets were pickedand aliquoted at room temperature into batches of five islets inKrebs-Ringer bicarbonate buffer (KRB; 16.7 mM glucose, 5 mM HEPES, 0.2%RIA grade BSA). Islets were picked directly into 12×75 mm glass tubeswith the bottom of tube replaced with 62 micron polyester mesh. These“mesh baskets” allow for easy transfer of islets from preincubationmedia to incubation media with very little disturbance of the islets.

After the islets were aliquoted into the mesh baskets, they were placedin 1 ml preincubation media, KRB containing 1.7 mM glucose, 0.5% BSA, at37 degrees for 45 minutes. Each tube was gassed with 95% oxygen/5%carbon dioxide, and then capped to maintain pH at 7.4. At the end of the45 minute preincubation islets in mesh baskets were gently transferredto the incubation media, 1 ml KRB containing 0.5% BSA and theappropriate test agents (glucose concentration was determined by testagents used, but the minimal glucose concentration was 1.7 mM).Incubation period was for 45 minutes at 37 degrees; again each tube wasgassed and capped for the duration of the incubation period. Thisculture is referred to herein as a “glucose culture.”

At the end of the incubation period, the mesh basket was removed. Theincubation media was frozen at −20 and assayed for insulin using theLinco RIA kit. Islets in the mesh basket were placed into 1 ml acidethanol for the extraction of islet insulin content. Extracted insulinwas diluted with RIA buffer and also assayed for insulin using the LincoRIA kit.

FIG. 3 depicts fractional insulin release from (a) a cultured Ins-1insulinoma cell line selected for high insulin secretion response in thepresence of glucose (see Hohmeier et al., Diabetes, 49:424 (2000)). FIG.3 demonstrates in a cell in which the effect of glucose is amplified,that DPD at the indicated concentrations can have a marked positiveeffect upon insulin secretion in the presence of low (3 mM) or high (15mM) glucose. The cells were incubated at 3 mM glucose with the indicatedconcentrations of either αKG or the DPD analog for 2 hours. Then, thecells were incubated for an additional 2 hours in either 3 or 15 mMglucose.

In a similar trial, FIG. 4 depicts fractional insulin release from thecells used in FIG. 3 as well as from a commonly used rat Ins-1 beta cellline (see Asfari et al., Endocrinology 130:167 (1992)). For comparison,while the data from rat Ins-1 cells, shown at the right side of FIG. 4,also evidence an increase in insulin secretion in the presence of DPD,the effect is not as dramatic, thereby demonstrating the desirability ofemploying a responsive cell line when analyzing the effects αKG and itsanalogs on insulin secretion.

PROPHETIC EXAMPLE

Intact pancreatic β cells are treated with compounds that promoteinsulin secretion by suppressing efflux of potassium ions throughK_(ATP). Depolarization of the resting membrane potential is observedusing micro-electrodes to monitor whole cell voltage, causingvoltage-gated calcium ion channels to open, leading to an influx ofcalcium ions and triggering insulin secretion.

Intact pancreatic β cells are treated with compounds that promoteinsulin secretion by suppressing efflux of potassium ions throughK_(ATP) and with a modulator of K_(ATP) such as diazoxide and glyburide.In the presence of the modulators, insulin secretion is reduced orprevented relative to the secretion from cells not treated with themodulator.

The present invention is not limited to the preceding examples anddisclosure, but rather embraces all such modifications and variations ascome within the scope of the appended claims.

1. A method for treating Type II diabetes in a human or non-humananimal, the method comprising the steps of: administering a compoundaccording to the formula

wherein R¹ and R² are each independently a C₁₋₄ alkyl, in an amountsufficient to increase glucose-induced insulin secretion; and observingan increase in insulin secretion by pancreatic β cells, whereby theincrease is characteristic of increased sensitivity to glucose-inducedinsulin secretion.
 2. The method of claim 1, wherein R¹ and R² are eachindependently a methyl group or an ethyl group.
 3. The method as claim2, wherein both R¹ and R² are a methyl group or both R¹ and R² are anethyl group.
 4. The method of claim 3, wherein the compound administeredhas the formula